Mechanism of pyocyanin abolishment caused by mvaT mvaU double knockout in Pseudomonas aeruginosa PAO1
Mechanism of pyocyanin abolishment caused by mvaT mvaU double knockout in Pseudomonas aeruginosa PAO1
复制标题
铜绿假单胞菌PAO1中mvaT mvaU双敲除导致绿脓素消除的机制。
DOI：
10.1080/21505594.2019.1708052
复制
发表时间：
2020-01-01
期刊：
影响因子：
5.2
通讯作者：
You, Xuefu
中科院分区：
文献类型：
--
作者：
Dong, Limin;Pang, Jing;You, Xuefu
MvaT和MvaU是H-NS家族的转录调控因子，绿脓菌素是铜绿假单胞菌产生的重要毒力因子。铜绿假单胞菌PAO 1的mvaT mvaU双敲除突变体在先前的研究中证明了绿脓菌素的消除。在这里，我们进一步探讨了机制。研究方向主要有两个：绿脓菌素生物合成途径和QS系统。通过启动子强度测定和Real-Time PCR检测，发现绿脓菌素生物合成基因的表达发生了显著变化，导致绿脓菌素产量降低;通过LC-MS/MS信号分子定量和Real-Time PCR检测，研究了绿脓菌素对QS系统的影响。在mvaT mvaU双敲除中，3-oxo-C12-HSL的产量明显增加，而C4-HSL和PQS的产量明显减少，这种变化可以通过mvaT或mvaU互补来恢复。与QS系统信号分子结合的转录激活因子基因的表达均降低，导致信号-转录激活因子复合物的形成减少。rhlR和pqsE的表达降低也导致phzA 1和phzA 2的表达降低。进一步研究发现，QS系统下调可能与QS系统阻遏物QsrO有关，其在mvaT mvaU双敲除后高度上调。抑制了绿脓菌素的合成，降低了生物膜的形成能力。这些结果也被转录组分析所证实，其显示了上述基因的类似基因表达变化以及QS系统调控的其他毒力因子基因的表达降低。
MvaT and MvaU are global transcriptional regulators belonging to the H-NS family, and pyocyanin is an important virulence factor produced by Pseudomonas aeruginosa. mvaT mvaU double knockout mutant of P. aeruginosa PAO1 demonstrated pyocyanin abolishment in the previous study. Here, we further explored the mechanism. Two main directions were studied: pyocyanin biosynthesis pathway and QS system. The effect on the expression of the pyocyanin biosynthesis genes was evaluated by promoter strength determination and Real-Time PCR assay, and significant changes leading to low pyocyanin production were found. The effect on the QS system was studied by signal molecule quantification using LC-MS/MS and related gene expression measurements using Real-Time PCR. In mvaT mvaU double knockout, the production of 3-oxo-C12-HSL obviously increased, while those of C4-HSL and PQS obviously decreased, and the changes can be recovered by mvaT or mvaU complementation. The expressions of transcriptional activator genes binding with QS system signal molecules were all decreased, resulting in decreased formation of signal-transcriptional activator complexes. And the decreased expression of rhlR and pqsE also led to the lower expression of phzA1 and phzA2. Further exploration found that QS system downregulation may be related to QsrO, a QS system repressor, which was highly upregulated with mvaT mvaU double knockout. Hence, the synthesis of pyocyanin was suffocated and the biofilm formation ability was decreased. These results were also confirmed by transcriptome analysis, which demonstrated similar gene expression changes of the aforementioned genes together with decreased expression of other virulence factor genes regulated by QS system.